Traumatic brain injury (TBI) is inevitably complicated by microvascular dysfunction and/or failure, resulting in formation of edema and, even worse, "progressive secondary hemorrhage" (PSH). These abnormalities lead to ischemia, extravasation of blood and inflammatory cells, autodestruction of CNS tissues, and worsening of neurological outcome. We recently discovered that the NCCa-ATP channel is critically involved in PSH, with strong evidence showing critical involvement of sulfonylurea receptor 1 (SUR1), the regulatory subunit of channel. New preliminary data strongly suggest that an unusual member of the transient receptor potential (TRP) family, TRPM4, acts as the pore-forming subunit of the channel, and that the channel is transcriptionally upregulated in microvascular endothelial cells via mechano-activation of NF-?B, which occurs within minutes of force delivery to tissues in vivo and to cultured endothelial cells in vitro. Our overarching hypothesis is that trauma-associated mechanical forces activate the mechano-sensitive transcription factor, NF-?B, resulting in transcriptional upregulation of TRPM4 channels in microvascular endothelium, and that these channels play a critical role in the pathophysiology of progressive secondary hemorrhage (PSH) following TBI. Numerous ramifications of this hypothesis will be tested using molecular and cell biology, and patch clamp electrophysiology. In Specific Aim (SA) 1, we will use TRPM4 gene knock-out mice to determine the extent to which TRPM4 channels contribute to the overall pathophysiology of PSH. In the same Aim, we will also evaluate 2 treatments with strong translational potential: brief (24-hr) TRPM4 gene suppression using IV antisense oligodeoxynucleotide (AS-ODN), and riluzole, a pleiotropic FDA-approved drug that is a potent blocker of TRPM4 channels. In SA2, we will examine physiological modulation of the channel in freshly isolated CNS capillaries from TBI-mice, and in cultured endothelial cells exposed to a "trauma-like" mechanical stimulus (stretch), and we will determine the specific role of the channel in dysfunction and death of capillary endothelial cells. In SA3, we will examine the role of the mechano-activated transcription factor, NF-?B, in transcriptional regulation of the channel, and we will assess whether targeting this transcription factor in TBI is beneficial due to interference with expression of TRPM4 channels. TBI continues to be a leading cause of death and disability, with no treatments yet available to reduce secondary injury. We expect to find that TRPM4 plays a critical role as end-executioner in capillary destruction leading to PSH following TBI, and that block of TRPM4 by gene suppression, at the transcriptional level, or pharmacologically will yield significant reductions in the multiple cascades of secondary injury attributable to PSH and hemorrhage. These findings would establish a novel and powerful therapeutic target in TBI, leading to further pre-clinical as well as clinical trials with AS-ODN and/or riluzole, which we predict will have a major impact on acute treatment of TBI in humans. PUBLIC HEALTH RELEVANCE: TBI continues to be a leading cause of death and disability, with no treatments yet available to reduce secondary injury. We expect to find that TRPM4 plays a critical role as end-executioner in capillary destruction leading to PSH following TBI, and that block of TRPM4 by gene suppression, at the transcriptional level, or pharmacologically will yield significant reductions in the multiple cascades of secondary injury attributable to PSH and hemorrhage. These findings would establish a novel and powerful therapeutic target in TBI, leading to further pre-clinical as well as clinical trials with AS-ODN and/or riluzole, which we predict will have a major impact on acute treatment of TBI in humans.